The objective of this proposal is to identify internalizing human single chain antibodies (scFvs) that target pancreatic tumor cells in situ, with emphasis on (1) early stage tumors (stages 0/I), for which internalizing scFvs can be used in non-invasive imaging for early detection, and (2) late stage tumors (stages III/IV), for which internalizing scFvs can be used to develop targeted therapy based on intracellular delivery strategies. Currently, there are very few human monoclonal antibodies that target pancreatic tumors, and even fewer that specifically detect early stage pancreatic tumor to allow non-invasive imaging of asymptomatic neoplasia, an area that has a huge impact on treatment options and outcomes. Thus our proposed research would address a critical need of this field. We hypothesize that like other tumors, pancreatic tumors possess unique cell surface epitope profiles that distinguish tumors from non-neoplastic tissues. Some of these epitopes may be present early in tumor progression, allowing early detection of asymptomatic neoplasia. We further hypothesize that a subset of these tumor cell surface epitopes are internalizing, and thus can be utilized to deliver payloads intracellularly to pancreatic tumor cells. Because the tumor cell surface epitope space is composed of complex antigenic determinants including posttranslational modifications, we have taken an antibody-based approach to study the tumor cell surface. We have previously developed and utilized a naive phage antibody library containing 500 million members to identify human single chain antibodies (scFvs) targeting tumor associated internalizing epitopes, facilitating further development of targeted therapy based on intracellular delivery strategies. Recognizing that tumor cell lines often express a different cell surface antigenic profile than that of tumor cells in situ, we have recently developed a novel method that utilizes laser capture microdissection (LCM) to select phage antibody libraries on tumor cells in situ, thereby generating antibodies against clinically represented tumor antigens as opposed to possible artifacts associated with cell lines cultured in vitro. By using these techniques we are able to identify human monoclonal antibodies that target tumor antigens expressed in situ in clinical specimens, and possess novel, therapeutically useful functions such as targeted intracellular payload delivery. The precise procurement of disease cells by LCM allows tumor cells at different stages to be targeted for phage antibody selection, offering opportunities to identify stage-associated antibodies that may be used in early tumor detection. We propose to apply this strategy to select internalizing human scFvs that target pancreatic tumor cells in situ at different stages. These novel antibodies, which are human in sequence, can in the future be used to develop targeted therapeutics and non-invasive imaging-based early tumor detection. PUBLIC HEALTH RELEVANCE: This project aims to identify by laser capture microdissection internalizing human single chain antibodies that target pancreatic tumor cells in situ. These internalizing scFvs can be used to develop non-invasive imaging strategies that allow sensitive and accurate early tumor detection, and targeted therapies based on intracellular delivery strategies.